SECONDARY METABOLITE CONGENER DISTRIBUTION MODULATION

Description

Field of the Invention

[0001] The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology, and methods of modulating polyketide congener distribution by modulating oxygen tension. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.

Background of the Invention

[0002] Polyketides represent a large family of diverse, compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; United States Patent Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; and 5,962,290; and Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888.

[0003] Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or "modular" and Type II "iterative" PKS enzymes. A third type of PKS found primarily in fungal cells has features of both the Type I and Type II enzymes and is referred to as a "fungal" PKS enzymes.

[0004] Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more "modules" of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or "domains." These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying B-carlion processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991).

[0005] During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Patent Nos. 5,672,491 and 5,712,146). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a "clean" host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication Nos. WO 98/49315 and 00/024907).

[0006] The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9:403-411; and U.S. Patent Nos. 5,712,146 and 5,672,491). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide, synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.

[0007] There has been a great deal of interest in expressing polyketides produced by Type I and Type II PKS enzymes in host cells that do not normally express such enzymes. For example, the production of the fungal polyketide 6-methylsalicylic acid (6-MSA) in heterologous E. coli, yeast, and plant cells has been reported. See Kealey et al., Jan. 1998, Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic host, Proc. Natl. Acad. Sci. USA 95:505-9, U.S. Patent No. 6,033,883, and PCT Patent Publication Nos. 98/27203 and 99/02669. Heterologous production of 6-MSA required or was considerably increased by co-expression of a heterologous acyl carrier protein synthase (ACPS) and that, for E. coli, media supplements were helpful in increasing the level of the malonyl CoA substrate utilized in 6-MSA biosynthesis. See also, PCT Patent Publication No. 97/13845.

[0008] The biosynthesis of other polyketides requires substrates other than or in addition to malonyl CoA. Such substrates include, for example, propionyl CoA, 2-methylmalonyl CoA, 2-hydroxymalonyl CoA, and 2-ethylmalonyl CoA. Of the myriad host cells possible for utilization as polyketide producing hosts, many do hot naturally produce such substrates. In addition, to the manipulation of starting substrates and extender molecules there are manipulations of tailoring enzymes in various hosts to achieve diverse polyketide products. Furthermore, the host plays a significant role in polyketide congener production.

[0009] Bacteria are classified as strict aerobes (oxygen is necessary for growth), facultative anaerobes (oxygen is not necessary, but results in faster growth), aero tolerant anaerobes (oxygen has no effect on growth), strict anaerobes (oxygen prevents growth), or as microaerophilic (low concentrations of oxygen are required for growth). Oxygen-dependent intracellular processes in aerobic bacteria are mediated by oxidase and oxygenase enzymes. Most myxobacteria and actinomycetes are examples of strict aerobes. Growth of aerobic bacteria under microaerobic (low oxygen) conditions is of interest, as the culture's growth behavior and production of primary and secondary metabolites is often different than that typically observed under a condition of excess oxygenation (Winkelheusen et al., 1996; Schneider et al. 1999; Jensen et al., 2001; Liefke et al.. 1990; Kaiser et al., 1994; Dick et al, 1994). References cited by author and year of publication are given a full citation below.

[0010] Many secondary metabolites produced by members of the Actinomycetales and Myxococcales exhibit potent and varied biological activities. Polyketides are a class of these natural products produced by large multifunctional enzymes called polyketide synthases (PKS), often followed by modification via a subsequent enzymatic pathway (Carreras et al., 2000). The actions of these tailoring pathway enzymes can include glycosylations, hydroxylations, methylations, epoxidations, or a number of other changes to the core structure of the molecule.

[0011] Intermediate compounds along these tailoring pathways often possess biological potencies different from the final compound. Examples of this are the antibacterial compounds erythromycins B, C, and D, which can be processed into the more potent erythromycin A congener via an enzymatic pathway which includes the oxygen-dependent EryK hydroxylase (see Figure 1). Erythromycin B and its 13-substituted analogs are useful as precursors for the semisynthetic production of prokinetic gastrointestinal agents called motilides. However, erythromycin B is a very poor substrate for the EryK enzyme, and is essentially a shunt end product of this pathway (Lambalot et al., 1995). The ultimate fate of an erythromycin D molecule is then dependent upon a competitive reaction between the EryG methylase and the oxygen dependent EryK hydroxylase. It has been shown previously that the dissolved oxygen concentration during S. erythraea bioconversions of 6-deoxyerythronolide B (6-dEB) analogs is critical in determining if the erythromycin A or B congener is the predominant end product of these cultivations (Carreras et al., 2002).

[0012] The epothilones (Figure 2) have recently generated interest as potential chemotherapeutic successors to the potent anticancer compound paclitaxel (Bollag et al., 1995, Gerth et al., 1996). Both paclitaxel (Taxol^®) and the epothilones stabilize microtubules via the same mechanism of action, but the epothilones are effective against paclitaxel-resistant tumors and are more water-soluble (Kowalski et al., 1997, Su et al., 1997). At least 39 different epothilone variants and related compounds have been identified in the fermentation broth of the wild type producing organism, Sorangium cellulosum (Hardt et al., 2001). Epothilone D is reported to possess the highest therapeutic index of the four major epothilone congeners A, B, C, or D (Chou et al., 1998, Chou et al., 2001), but is produced in very low quantity by S. cellulosum (Gerth et al., 2000). The acyl transferase 4 (AT4) domain of the epothilone PKS can incorporate either a malonyl-CoA or amethylmalonyl-CoA extender unit, with this selectivity influencing the final ratio between epothilones A and B, or between epothilones C and D (Gerth et al., 2000). The epothilone D congener is a direct precursor for epoxidation into epothilone B (Figure 3), catalyzed by the EpoK monooxygenase (Julien et al., 2000, Gerth, et al., 2001). WO 01/83800 describes recombinant host cells of the suborder Cystobacterineae and the genus Myxococcus which contain recombinant expression vectors that encode heterologous PKS genes. These cells are described as useful in the production of epothilones and other polyketides.

[0013] For example, WO 01/83800 reports the production of epothilone D by Myxococcus xanthus strain K111-40.1 expressing epothilone PKS genes and with a non-functional epoK gene, with dissolved oxygen controlled at 50% or greater than 80% of saturation. WO 01/83800 also reports the production of epothilone B by Myxococcus xanthus strain K111-32.25 expressing epothilone PKS genes and with a functional epoK gene, with dissolved oxygen at greater than 80% of saturation.

[0014] Erythromycin B and epothilone D are presented as examples of intermediates of secondary metabolite tailoring pathways in which the intermediate is sometimes the desired product. Additionally, there are many other examples of natural products in which a monooxygenase enzyme reaction is necessary for processing of a pathway intermediate into the end product. Examples include the bioconversion of compactin into the cholesterol-lowering drug, pravastatin (Serizawa et al., 1991, Watanabe et al., 1995; the production of the plant hormone, gibberellin (Tudzynski et al., 1998); the production of a fungal mycotoxin, sterigmatocystin (Keller et al., 2000); and the production of doxorubicin, an anti-cancer agent (Lomovskaya et al., 1999, Walczak et al. 1999).

[0015] The activity of these oxygen-dependent enzymes can be eliminated by genetic means, but this engineering may prove difficult or impossible to accomplish for various reasons. Directed genetic inactivation of a monooxygenase enzyme requires knowledge of and access to its sequence, as well as the ability to manipulate the DNA of the host organism. The present invention provides an alternate method for producing these intermediates as the major products, without requiring any genetic manipulation of the tailoring enzymes.

[0016] Given the potential for making valuable and useful polyketides in large quantities in heterologous host cells, there is a need for host cells capable of making polyketides in the modified distribution of congeners. The present invention helps meet that need by providing recombinant host cells, expression vectors, and methods for making polyketides in diverse host cells.

[0017] The following references provide background information on the present invention.

Winkelhausen, E.; Pittman, P.; Kuzmanova, S.; Jeffries, T. Xylitol formation by Candida boidinii in oxygen limited chemostat culture. Biotechnol. Lett. 1996, 18, 753-758.

Schneider, S.; Wubbolts, M.; Oesterhelt, G.; Sanglard, D.; Witholt, B. Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations. Biotechnol. Bioeng. 1999, 64, 333-341.

Jensen, N.; Melchiorsen, C.; Jokumsen, K.; Villadsen, J. Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate. Appl. Environ. Microbiol. 2001, 67, 2677-2682.

Liefke, E.; Kaiser, D.; Onken, U. Growth and product formation of actinomycetes cultivated at increased total pressure and oxygen partial pressure. Appl. Environ. Microbiol. 1990, 32, 674-679.

Kaiser, D.; Onken, U.; Sattler, I.; Zeeck, A. Influence of increased dissolved oxygen concentration on the formation of secondary metabolites by manumycin-producing Streptomyces parvulus. Appl. Environ. Microbiol. 1994, 41, 309-312.

Dick, O.; Onken, U.; Sattler, I.; Zeeck, A. Influence of increased dissolved oxygen concentration on productivity and selectivity in cultures of a colabomycin-producing strain of Streptomyces griseoflavus. Appl. Environ. Microbiol. 1994, 41, 373-377.

Can-eras, C.; Ashley, G.W. Manipulation of Polyketide Biosynthesis for New Drug Discovery. In New Approaches to Drug Development; Jolles, P., Ed.; Birkhauser-Veriag, Switzerland, 2000; pp 89-108.

Lambalot, R.H.; Cane, D.E.; Aparicio, J.J.; Katz, L. Overproduction and characterization of the erythromycin C-12 hydroxylase, EryK. Biochemistry 1995, 34, 1858-1866.

Carreras, C.; Frykman, S.; Ou, S.; Cadapan, L.; Zavala, S.; Woo, E.; Leaf, T.; Carney, J,; Burlingame, M.; Patel, S.; Ashley, G.; Licari, P.J. Saccharopolyspora erythraea-catalyzed bioconversion of 6-deoxyerythronolide B analogs for production of novel erythromycins. J. Biotechnol. 2002, 92, 217-228.

Bollag, D.M.; McQueney, P.A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C.M. Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res. 1995, 55, 2325-2333.

Gerth, K.; Bedorf, N.; Höfle, G.; Irschik, H.; Reichenbach, H. Epothilons A and B: Antifungal and cytotoxic compounds from Sorangium cellulosum (myxobacteria)- production, physico-chemical, and biological properties. J. Antibiot. 1996, 49, 560-563.

Kowalski, R.J.; Giannakakou, P.; Hamel, E. Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel. J. Biol. Chem. 1997, 272, 2534-41.

Su, D.S.; Meng, D.; Bertinato, P.; Balog, A.; Sorensen, E.J.; Danishefsky, S.J.; Zheng, Y.H.; Chou, T.C.; He, L.; Horwitz, S.B. Structure-activity relationships of the epothilones and the first in vivo comparison with paclitaxel. Angew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096.

Hardt, L; Steinmetz, H.; Gerth, K.; Sasse, F.; Reichenbach, H.; Höfle, G. New natural epothilones from Sorangium cellulosum, strains So ce90/B2 and So ce90/DI3: Isolation, structure elucidation, and SAR studies. J. Nat. products. 2001, 64, 847-856.

Chou, T.C.; Zhang, X.G.; Balog, A.; Su, D.S.; Meng, D.; Savin, K.; Bertino, J.R.; Danishefsky, S.J. Desoxyepothilone B: An efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B. Proc. Natl. Acad. Sci. USA 1998, 95, 9642-9647.

Chou, T.C.; O'Connor, O.A.; Tong, W.P.; Guan, Y.; Zhang, Z.; Stachel, S.J.; Lee, C.; Danishefsky, S.J. The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice. Proc. Natl. Acad. Sci. USA 2001, 98, 8113-8118.

Gerth, K.; Steinmetz, H.; Höfle, G.; Reichenbach, H. Studies on the biosynthesis of epothilones: the biosynthetic origin of the carbon skeleton. J. Antibiot. 2000, 53, 1373-1377.

Julien, B.; Shah, S.; Zierman, R.; Goldman, R.; Katz, L.; Khosla, C. Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum. Gene. 2000, 249, 153-160.

Gerth, K.; Steinmetz, H.; Höfle, G.; Reichenbach, H. Studies on the biosynthesis of epothilones: The PKS and epothilone C/D monooxygenase. J. Antibiot. 2001, 54, 144-148.

Serizawa, N.; Matsuoka, T. A two component-type cytochrome P-450 monooxygenase system in a prokaryote that catalyzes hydroxylation of ML-236B to pravastatin, a tissue-selective inhibitor of 3-hydroy-3-methylglutaryl coenzyme A reductase. Biochem. Biophys. Acta. 1991, 1084, 35-40.

Watanabe, 1.; Nara, F.; Serizawa, N. Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene. 1995, 163, 81-85.

Tudzynski, B.; Holter, K. Gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence of a gene cluster. Fungal Genet. BioL 1998, 25, 157-170.

Keller, N.; Watanabe, C.; Kelkar, H.; Adams, T.; Townsend, C. Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans. Appl. Environ. Microbiol: 2000, 66, 359-362.

Lomovskaya, N.; Otten, S.; Doi-Katayama, Y.; Fonstein, L.; Liu, X.; Takatsu, T.; Inventi-Solari, A.; Filippini, S.; Torti, F.; Columbo, A.; Hutchinson, C.R. Doxorubicin overproduction in Streptomyces peucetius: cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene. J. Bacteriol. 1999, 181, 305-318.

Walczak, R.; Dickens, M.; Priestley, N.; Strohl, W. Purification, properties, and characterization of recombinant Streptomyces sp. Strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis. J. Bacteriol. 1999, 181, 298-304.

Julien, B.; Shah, S. Heterologous expression of the epothilone biosynthetic genes in Myxococcus xanthus. Antimicrob. Agents Chemother. 2002, 46, 2772-2778

Pirt, S. J. Oxygen Demand and Supply. In Principles of Microbe and Cell Cultivation. John Wiley and Sons, New York, 1975; pg. 84.

Lau, J.; Frykman, S.; Regentin, R.; Ou, S.; Tsuruta, H.; Licari, P. Optimizing the heterologous production of epothilone D in Myxococcus xanthus. Biotechnol. Bioeng. 2002, 78, 280-288.

Arras, T.; Schirawski, J.; Unden, G. Availability of O2 as a substrate in the cytoplasm of bacteria under aerobic and microaerobic conditions. J. Bacteriol. 1998, 180, 2133-2136.

Tang, L.; Shah, S.; Chung, L.; Carbey, J.; Katz, L.; Khosla, C.; Julien, B. Cloning and heterologous expression of the epothilone gene cluster. Science 2000, 287, 640-642.

Frykman, S.; Tsuruta, H.; Lau, J.; Regentin, R; Ou, S; Reeves, C.; Carney, J.; Santi, D.; Licari, P. Modulation of epothilone analog production through media design. J. Ind. Microbiol. Biotechnol. 2002,28, 17-20.

Summary of the Invention

[0018] Described herein is a method of modulating the polyketide congener distribution in a heterologous host cell without genetic manipulation of the polyketide synthase (PKS) genes or tailoring enzymes. In particular, the present invention provides a method of modulating polyketide congener distribution production ratio in a myxobacterial host cell that comprises an active oxygen-dependent tailoring enzyme, the method comprising the steps of cultivating said host cell under depleted oxygen cultivation in growth medium under conditions that lead to the production of polyketides, wherein the production ratio of polyketide congeners is shifted from epothilone A and B to epothilone C and D.

[0019] Described herein are metabolically engineered heterologous host cells that can be manipulated to produce a certain distribution of polyketide congeners by controlling oxygen tension during cultivation of said host cells.

[0020] Also described herein are recombinant polyketide producing host cells that when cultivated under modulated oxygen tension conditions shift production of congeners. The recombinant host cells may be Sorangium cellulosum host cells that produce epothilones A and B and shift production to epothilone C and D when cultivated under depleted oxygen conditions.

[0021] The polyketide produced by the heterologous host cell may be epothilone, and the epothilone congener distribution may be modulated to produce epothilone D by modulating the oxygen tension during cultivation.

[0022] The epothilone congener distribution modulated by oxygen tension may result in a higher ratio of epothilone D congener to epothilone C congener under excess oxygen tension. The ratio of epothilone C to epothilone D congeners may increase under depleted oxygen conditions. The epothilone congeners may be produced by a heterologous Myxococcus xanthus strain K111-32.25. Epothilones A and B may be produced under excess oxygen tension, and epothilones C and D may be produced under depleted oxygen tension in strain K111-32.25.

[0023] Described herein is a method of altering the specificity of acyltransferase (AT) domains of epothilone by modulating oxygen tension of module 4 of epothilone polyketide synthase (PKS) cluster without genetic manipulation. The AT specificity of strain K111-32.25 may be shifted to methylmalonyl-CoA resulting in a shift towards congeners with methyl group at C-12 (epothilones B or D).

[0024] Also described herein are novel polyketides produced by metabolically engineered heterologous host cells having an active EpoK monooxygenase.

[0025] The novel polyketide produced by modulating oxygen tension during cultivation of a heterologous host cell may be epothilone 506.

Brief Description of the Drawings

[0026] Figure 1 shows erythromycins B, C, and D, which can be processed into the more potent erythromycin A congener via an enzymatic pathway which includes the oxygen-dependent EryK hydroxylase.

[0027] Figure 2 shows the chemical structure of various epothilones. (a) Epothilone A (R₁ = H; R₂ = S); Epothilone B (R₁ = CH₃; R₂ = S); Epothilone G₁ (R₁ = H; R₂ = O); Epothilone G₂ (R₁ = CH₃; R₂ = O). (b) Epothilone C (R₁ = H; R₂ = S); Epothilone D (R₁ = CH₃; R₂ = S); Epothilone H₁ (R₁ = H; R₂ = O); Epothilone H₂ (R₁ = CH₃; R₂ = O). (c) 10,11-didehydro-epothilone A (R₁ = H); 10,11-didehydro-epothilone B (R₁ = CH₃). (d) 10,11-didehydro-epothilone C (R₁ = H); 10,11-didehydro-epothilone D (R₁ = CH₃).

[0028] Figure 3 shows epothilone D congener is a direct precursor for epoxidation into epothilone B catalyzed by the EpoK monooxygenase.

[0029] Figure 4 shows cultivation of the strain K111-32.25 (EpoK⁺) under (a) excess or (b) depleted oxygenation, with depleted oxygenation resulting in a 60% higher cell density than that obtained during cultivation of the same strain in the presence of excess oxygen.

[0030] Figure 5 shows HPLC chromatographs of resin extracts from fermentations of strain K111-40.1 (EpoK⁺) cultivated under (a) excess oxygen condition resulting in maximal activity of the EpoK enzyme, and production of epothilones A and B as the primary final products; (b) depleted oxygen condition limits the activity of this enzyme; resulting in production of epothilones C and D as the primary final products; (c) result of these cultivations under depleted oxygenation is similar to those obtained with another strain (K111-40.1) under excess oxygenation in which the activity of the EpoK enzyme has been eliminated through genetic engineering.

[0031] Figure 6 shows vinylogous Payne rearrangement of (a) 10,11-didehydro-epothilone B converts to (b) Epo506 under excess oxygen conditions.

[0032] Figure 7 shows cultivation of strain K165-79.7 under both depleted and excess oxygenation conditions resulting in shifting of the congener ratio between 10,11-didehydro-epothilone D and Epo506.

[0033] Figure 8 shows modulation of oxygen tension during cultivation of Sorangium cellulosum strain Soce K111-150.17 shifts epothilone product distribution. (a) cell growth under 50 % dissolved oxygen (DO); and (b) and (c) low oxygen tension.

[0034] Figure 9 shows titer time course for three different dissolved oxygen conditions.

[0035] Figure 10 shows an HPLC chromatogram showing epo A and epo B congener distribution from 50% dissolved oxygen condition at t=112 hrs.

[0036] Figure 11 shows an HPLC chromatogram of epothilones A, B, C, and D peaks from the 0% dissolved oxygen condition at 112 hrs.

Detailed Description of the Invention

[0037] The present invention provides an alternate method for producing polyketide intermediates as the major products, without requiring any genetic manipulation of the tailoring enzymes. The present methods of the present invention utilize host cells that have been deposited with the American Type Culture Collection (ATCC) under the Budapest Treaty and assigned deposit designation numbers. Myxococcus xanthus strain K111-32.25 was deposited May 1, 2000 under deposit designation PTA-1700, and Myxococcus xanthus strain K111-40.1 was deposited January 18, 2001 under deposit number FTA-2712.

[0038] A fed batch cultivation process has been previously described for the production of epothilone D in heterologous M. xanthus system. During these fed-batch cultivations, the dissolved oxygen tension can be modulated or regulated from excess oxygen, about 50% dissolved oxygen saturation to depleted oxygen or 0% dissolved oxygen measured, to achieve shifting of congener production. During fed-batch cultivations, the dissolved oxygen tension is maintained at 50% of air saturation through automated control of the agitation rate. When the upper range of the agitation rate is limited at a value insufficient for maintenance of this dissolved oxygen tension, continued increase of the culture's metabolic demand by its increasing cell density can eventually deplete the cultivation broth of oxygen. The culture is then in a metabolic state in which the rate of oxygen dissolution into the culture broth is equivalent to its consumption rate by the cells. This metabolic state results in a reading of zero from the dissolved oxygen probe, indicating that no residual oxygen activity is detected in the culture broth. This culture state can then be exploited to minimize the activity of oxygen-dependent tailoring enzymes of the processing pathway, while still providing enough oxygen to maintain culture viability.

[0039] Cultures of the K1 11-32.25 (ATCC deposit designation PTA-1700, May 1, 2000) epothilone B producing strain (EpoK⁺) were maintained at excess (50% of saturation) and depleted (0%) dissolved oxygen tensions during the entire epothilone production phase by controlling the agitation rate (Figure 4 a,b). The excess oxygen condition results in maximal activity of the EpoK enzyme, and production of epothilones A and B as the primary final products (Figure 5a). The depleted oxygen condition limits the activity of this enzyme, resulting in production of epothilones C and D as the primary final products (Figure 5b). The result of these cultivations under depleted oxygenation is similar to those obtained with' another strain (K111-40.1. ATCC deposit designation PTA-2712, January 18, 2001) under excess oxygenation in which the activity of the EpoK enzyme has been eliminated through genetic engineering (Figure 5c).

[0040] The modulation of the dissolved oxygen tension influences the ratio between the epothilone A and B (or C and D) congeners, in addition to the maximum total titer of the epothilones (See Example 2, Table 1). However, the time at which these maximum titers were attained was not dependent on the strain type or level of oxygenation in the bioreactor, as all three of the cultivation conditions described in Table 1 attained their maximum total epothilone titer around day 12. The acyl transferase 4 (AT4) domain of the epothilone PKS can incorporate either a malonyl-CoA or a methylmalonyl-CoA extender unit, with this selectivity influencing the final ratio between these epothilone congeners (17). The oxygen concentration may have an indirect influence on the intracellular pool distribution between malonyl- and methylmalonyl-CoA. Cultivation of strains K111-40.1 (EpoK^-) and K111-32.25 (EpoK⁺) with excess oxygenation resulted in a pronounced shift towards the congener with the methyl group at C-12 (epothilones B or D), indicating that the cells favor production and/or incorporation of methylmalonyl-CoA under this condition (Table 1).

[0041] Cultivation of the EpoK⁺ strain with depleted oxygenation resulted in a cell density that was 60% higher than that obtained during cultivation of the same strain in the presence of excess oxygen (Figure 4 a,b). The exponential growth phase of the excess oxygen cultivation ceased on day 7, after which the cell density remained relatively constant (Figure 4a). The specific growth rate (µ) during this exponential phase was 0.7 d^-1. However, when the dissolved oxygen tension in the depleted oxygen cultivation reached zero on day 4 due to limitation of the agitation cascade response, this culture shifted from its exponential growth phase to a slower rate of growth (µ = 0.15 d^-1), which continued until day 12 (Figure 4b). A similar pattern in growth behavior and increase in the maximum cell density was also observed with the EpoK- strain with depleted oxygenation, as well as with other genetically engineered M. xanthus strains which produce novel epothilones.

[0042] The increase in the cell density in the depleted oxygen cultivations can be attributed to (i) the cells may utilize nutrients at a different rate or in a different selective pattern during the slow growth imposed by oxygen limitation, thereby postponing the exhaustion of critical nutrients, (ii) the cultures produce different types or levels of potentially growth-inhibitory metabolites during oxygen limitation than excreted under excess oxygenation, or (iii) limitation of oxygen enhances or represses the expression of genes and the activity of enzymes involved in bacterial growth and respiration processes.

[0043] Control of the dissolved oxygen concentration during cultivation of bacterial cultures can have a dramatic effect on the cell density of the culture, as well as secondary metabolite titers and their congener distributions. This oxygen-limited cultivation strategy can be used to produce the intermediates of an oxygen-dependent tailoring pathway as the primary products. However, monooxygenase activity is not completely eliminated, but merely reduced to a fraction of the maximal rate typically seen under excess oxygen conditions. To prevent these intermediates from still being processed at a slower rate, it may be necessary for them to be stabilized in some manner. An example of this is the competitive reaction between the EryK hydroxylase and the EryG methylase for the erythromycin D substrate (Figure 1). If the activity of EryK is reduced, EryG is able to catalyze, conversion of erythromycin D into erythromycin B, a poor substrate for the EryK hydroxylase. This causes accumulation of the enzymatic pathway intermediate, erythromycin B. This pathway also contains another oxygen-dependent tailoring enzyme, EryF (Figure 1), which remains capable of processing 6-deoxyerythronolide B (6-dEB) into erythronolide B under limited oxygen conditions.

[0044] Another example of this intermediate accumulation is the production of epothilone D during cultivation of a heterologous M. xanthus system with an active EpoK enzyme. The reduction in activity of an oxygen-depleted EpoK enzyme is apparently sufficient to slow the catalyzation of the epothilone D intermediate into its epothilone B congener and permit epothilone D to diffuse out of the cell and be bound and stabilized by the XAD-16 resin in the bioreactor. It has been shown that addition of epothilones C and D to shake flask cultures of a nonproducing strain of S. cellulosum which retains EpoK activity results in the diffusion of these compounds back into the cells and their transformation into epothilones A and B when no resin is present. Julien and coworkers have shown that this is also the case with a Myxococcus xanthus strain in which the epothilone PKS is nonfunctional, but EpoK is active.

[0045] Novel epothilones are created by strains in which the epothilone polyketide synthase sequence has been manipulated (Julien et al., 2000, Tang et al., 2000). Modulation of the dissolved oxygen tension, in combination with the addition of specific precursors such as acetate and propionate to the production medium, permits the production of each of the four major epothilone congeners A, B, C, or D (Figure 2) as the primary product from a single M. xanthus strain with an active EpoK enzyme (Frykman et al., 2002).

[0046] Increasing the serine concentration in the production medium increases its relative incorporation by the epothilone nonribosomal peptide synthetase (NRPS) into epothilone in place of cysteine (Frykman et al., 2002). Supplementation of serine to the production medium of strain K111-32.25, in conjunction with modulation of the dissolved oxygen concentration, results in the production of either of the oxazole epothilones G₂ or H₂ (Figure 2) as the major product. Supplementation of serine, acetate, and/or propionate to the production medium, in addition to control of the dissolved oxygen tension permits the accumulation of any of the eight thiazole and oxazole major compounds (epothilones A, B, C, D, G₁, G₂, H₁, and H₂) as the primary product from a single M. xanthus strain. This approach should also permit the production and congener modulation of the oxazole counterparts of 10,11-didehydro-epothilone D and Epo506, as well.

[0047] The wild type epothilone PKS has been shown to produce nearly 35 additional epothilone variants in addition to epothilones A - D (Hardt et al., 2001). Similarly, it has been found that heterologous expression of the epothilone PKS and engineered variants results in a number of novel compounds in addition to the expected product. The concentration of these products can be enhanced or suppressed by the cultivation conditions. The ability to generate new compounds and modulate their distribution through manipulation of the cultivation conditions of a strain possessing a single genetic change in its PKS sequence demonstrates how powerful a tool PKS engineering, when combined with suitable physiological tuning, can be in the rapid generation of new natural products with potent biological activities.

[0048] A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention.

Example 1

Cultivation of Host Cells Under Modulated Oxygen Tension Conditions

[0049] Bacterial Strains. The epothilone polyketide synthase was cloned from Sorangium cellulosum SMP44, and introduced into Myxococcus xanthus strain DZ1 (Julien et al., 2002). The M. xanthus epothilone B producer (K111-32.25) has a functional EpoK epoxidase (EpoK⁺), while this enzyme has been genetically inactivated (EpoK^-) in the epothilone D producing strain (K111-40.1). The activity of the enoyl reductase (ER5) domain of the epothilone PKS was eliminated by introduction of a point mutation in both the K111-32.25 and K111-40.1 strains to produce strains K165-79.7 and K165-76.2, respectively. Strains K165-79.7 and K165-76.2 were engineered to produce 10,11-didehydro-epothilones B and D (Figure 2), respectively.

[0050] Bioreactor Inoculum Expansion. All media components were obtained from Sigma (St. Louis, MO), unless otherwise specified. All M. xanthus cultures were initiated by reviving a 1 mL frozen cell bank vial in a sterile 25 mL glass tube containing 3 mL of CYE-MOM media (10 g/L of Casitone, a pancreatic digest of casein (Difco, Detroit, MI), 5 g/L yeast extract (Difco), 1 g/L MgSO₄·7H₂O (EM Science, Gibbstown, NJ), and 2 mL/L of methyl oleate (Ernerest 2301, Cognis Corporation, Cincinnati, OH)) for two days at 28°C and 175 rpm. The entire contents of this tube were expanded for one day in 50 mL of CYE-MOM in a 250 mL baffled Erlenmeyer flask, and then this flask was used to inoculate 500 mL of CYE-MOM in a 2.8 L baffled Fernbach flask cultured for one day.

[0051] Fed-batch Bioreactor Cultivation. One hundred grams of XAD-16 hydrophobic resin (Rohm and Haas, Philadelphia, PA) and 10 g MgSO₄·7H₂O were combined in 4.5 L of deionized water, and sterilized (90 minutes, 121°C) in a 5 L bioreactor (B. Braun, Allentown, PA). This resin is incorporated into the production medium to bind the epothilones and stabilize them from degradation. Two separate feed loops of a 250 g/L solution of the casein digest and methyl oleate were connected to the bioreactor, and a sufficient quantity of these two nutrients added to the bioreactor to bring the initial casein digest and methyl oleate concentrations to 5 g/L and 2 mL/L, respectively. Finally, 25 mL of a 0.22 µm filter-sterilized trace elements solution (1% (v/v) concentrated H₂SO₄, 14.6 g/L FeCl₃·6H₂O, 2.0 g/L ZnCl₃, 1.0 g/L MnCl₂.4H₂O, 0.43 g/L CuCl₂·2H₂O, 0.31 g/L H₃BO₃, 0.24 g/L CaCl₂·6H₂O, and 0.24 g/L Na₂MO₄·2H₂O) was added aseptically. The bioreactor was then inoculated (5% v/v) with the CYE-MOM seed culture, with the casein digest (2 g/L/day) and methyl oleate (3, mL/L/day) feeds initiated 48 hours following inoculation. Airflow was held constant in the bioreactor at 0.4 vvm (airflow volume per vessel volume per minute).

[0052] The dissolved oxygen tension in the bioreactor was monitored using an in situ polarographic oxygen sensor. This probe was calibrated at a 0% dissolved oxygen tension by equilibrating the production medium with 100% nitrogen, and at a 100% dissolved oxygen tension by equilibrating the production medium with air. The initial agitation rate was set at 400 rpm, with no additional bioreactor, backpressure used during the cultivations. The saturation concentration of oxygen is approximately 7.7 mg/L at 28°C (Pirt, S.J., 1975). The dissolved oxygen tension was maintained at a minimum of 50% of air saturation (3.9 mg/L O₂) by an agitation cascade in the excess oxygen cultivations, with the agitation rate limited to a maximum of 450 rpm in the, depleted oxygen cultivations. The pH set point of 7.4 was maintained by automated addition of 2.5N H₂SO₄ and 2.5N KOH. The bioreactors were sampled daily for 12 - 14 days, followed by harvest of the resin.

[0053] Quantitation of Cell Density and Epothilone Titers. A 50 mL culture sample was removed daily from the bioreactor into a conical tube, and the broth decanted from the resin. A 1 mL aliquot of the broth was centrifuged in a 1.5 mL eppendorf tube at 12000g for 5 minutes, and the supernatant removed by pipet. The cell pellet was resuspended with deionized water to a total volume of 1 mL, and the absorbance of this cell suspension measured spectrophotometrically at 600 nm (Genesys 20, Thermo Spectronic, Rochester, NY). Myxococcus xanthus cultures grow as discrete unicellular rods, facilitating accurate optical density (OD) measurements. An OD measurement of 1.0 absorbance units correlates to a dry cell weight of approximately 0.3 g/L, with this correlation linear within the range of 0 to 40 OD units.

[0054] The XAD-16 resin was washed once with 45 mL of deionized water, again followed by its decanting. The resin was extracted in 25 mL of reagent-grade methanol for 30 minutes, and the epothilone titers quantitated by either HPLC or LC/MS. HPLC analysis was performed using a Hewlett Packard 1090 HPLC (Palo Alto, CA) with UV detection at 250 nm. A 50 uL aliquot of the methanol-extracted solution was injected across two 4.6 x 10 mm guard columns (MetaChem Inertsil C18 ODS 3, 5 µm, Ansys, Lake Forest, CA), and a longer column of the same material for chromatographic separation (4.6 x 150 mm). The method was isocratic with 70% acetonitrile and 30% water over a 12 minute run. LC/MS analysis was conducted on the same samples by injection into a PE Sciex API100LC (Perkin-Elmer, Shelton, CT), using an APCI source and the same guard and chromatographic columns as previously described. The chromatographic method was an acetonitrile/water gradient increased linearly from 35% to 100% over a ten-minute period.

Example 2

Production of Novel Epothilones

[0055] The epothilone PKS has been genetically altered to create strains of the present invention which produce novel epothilones which potentially may exhibit increased water solubility and/or potency against tumor cell lines. The oxygenation control method of the present invention was applied to an engineered Myxococcus xanthus production strain (strain K165-79.7, EpoK⁺) which had been genetically engineered to produce a hovel epothilone, 10,11-didehydro-epothilone B (Figure 2). When its counterpart, strain K165-76.2 (EpoK^-), was cultivated under an excess oxygenation condition, 10,11-didehydro-epothilones C and D (Figure 2) were found to be the sole products. The active EpoK enzyme in strain K165-79.7 was expected to catalyze epoxidation of this 10,11-didehydro-epothilone D intermediate into 10,11-didehydro-epothilone B (Figure 6a) in the presence of excess oxygen. However, this compound was not detected; instead, a novel isomer (Epo506) was the primary product of these cultivations (Figure 6b). High resolution mass spectrometry and one- and two-dimensional NMR were utilized to establish the structure of this tetrahydrofuran product (see Example 1).

[0056] This observed result may be the product of a process whereby the 10,11-didehydro-epothilone B formed under these excess oxygen conditions, is then converted to Epo506 via a vinylogous Payne rearrangement, as summarized in Figure 6. This reaction is favorable due to the epoxide opening to yield a relatively stable allylic carbocation intermediate. Cultivation of strain K165-79.7 under both depleted and excess oxygenation conditions resulted in shifting of the congener ratio between 10,11-didehydro-epothilone D and Epo506 (Figure 7), which was similar to the congener shift between the epoxidated and non-epoxidated epothilone compounds obtained by cultivation of the parent strain, K111-32.25, under these two dissolved oxygen levels (Table 1).

[0057] A high-resolution mass spectrum of Epo506 was obtained with a Mariner TOF spectrometer (Applied Biosystems, Foster City, CA) configured with a turbo-ionspray source in positive ion mode. 1-D and 2-D NMR spectra of Epo506 were recorded at 300K with a DRX 400 spectrometer (Bruker, Billerica, MA) equipped with a QNP z-axis gradient probehead. Chemical shifts were referred to δ 7.26 and 77.0 for ¹H and ¹³C spectra, respectively.

[0058] The molecular formula of Epo506 was determined to be C₂₇H₃₉NO₆ by HREIMS measurement of the [M+H]⁺ peak at m/z 506.25887 (calculated for C₂₇H₄₀NO₆S 506.25709), indicating the incorporation of one oxygen atom with respect to 10,11-didehydro-epothilone D. The ¹³C spectrum indicated that Epo506 possessed one double bond less than 10,11-didehydro-epothilone D, suggesting that Epo506 contains an additional ring. HSQC, HMBC, COSY, and TOCSY data allowed unambiguous assignment of the carbon connectivity. The chemical shifts at carbons 3, 7, 10 and 13 indicated that all four of these carbons were bonded to oxygen atoms. Two of these oxygenated carbons were postulated to be part of a cyclic ether to account for the molecular formula. The hydroxyl protons at carbons 3 and 13 were visible in both the 1-D and 2-D spectra, and their HMBC and COSY correlations to neighboring atoms confirmed their assignments. Therefore, the cyclic ether must include carbons 7 and 10, indicating a tetrahydrofuran structure. Significant Na⁺ and K⁺ adducts in the mass spectrum also confirmed that the ion observed at 506.3 by LC/MS is truly the molecular ion, and not a fragment generated upon ionization of a hypothetical parent diol.

Table 1. Epothilone Congener shifts in K111-40.1 strain.

Cultivation Conditions (Strain, Oxygen Level)	Peak Epothilone, B Titer (mg/L)	Peak Epothilone D Titer (mg/L)	Total (A - D) Epothilone Titer (mg/L)	Epothilone B : A Congener Ratio	Epothilone D : C Congener Ratio
K111-32.25
(EpoK+)	48	3	60	5.8 : 1	3.6 : 1
Excess Oxygen
K111-32.25
(EpoK+)	3	19	34	2.5 : 1	1.7 : 1
Depleted Oxygen
K111-40.1
(EpoK-)	0	30	36	-	5.2 : 1
Excess Oxygen

Example 3

Modulated Epothilone Congener Production in Sorangium cellulosum strain Soce90 K111-150.17

[0059] Modulation of dissolved oxygen during cultivation of heterologous host cells has been shown to shift polyketide congener distribution. In this example, an epothilone producing strain was subjected to oxygen tension resulting in shifting of epothilone congener distribution when cultivated under oxygen depletion conditions. Sorangium cellulosum strain Soce90 K111-150.17 is a rifamycin-resistant mutant derived from Soce90 parent strain through UV mutagenesis (B. Julien, unpublished results). The cultivation methodologies and analytical methods used in this example were as done as described in Example 1 above.

[0060] Sorangium cellulosum strain Soce90 K111-150.17 is an epothilone A and B producer that has an active epoK gene. Figure 8 (a) illustrates the cell growth over time measured by optical density (OD) at 600 nm in three different dissolved oxygen (DO) conditions and airflow measured in 1pm (liters per minute). Figure 8(b) shows the effect of dissolved oxygen modulation on the epothilone product distribution. The Sorangium cellulosum strain Soce90 K111-150.17, an epothilone A and B producer, produces epothilones C and D at 0% depleted dissolved oxygen conditions. Table 2 shows the epothilone titers observed produced by the K111-150.17 strain under various oxygen tension conditions. The observed production of epothilone D is several fold higher under 0% depleted dissolved oxygen than at 50% excess dissolved oxygen for strain K111-150.17.

[0062] Table 2. Epothilone congener ratio shifts under depleted oxygen in strain K111-150.17.

DO conc.	LPM	Bv/v/m	EpoA (mg/L)	EpoB (mg/L)	EpoC (mg/L)	EpoD/mg/L
50%	1	0.2	13.3	6.54	0	0.08
0%	0.4	0.2	3.87	2.06	0.66	0.35
0%	0.3	0.15	3.04	1.71	0.4	0.32

[0061] Figure 9 shows a time course of epothilone A, B, C and D production by strain K111-150.17 in three different dissolved oxygen regimens showing higher epothilone D production under low oxygen tension regimens. HPLC chromatograms from the fermentations conducted under 50% dissolved oxygen tension and 0% oxygen tension show the almost exclusive epothilone A and B production at 50% oxygen tension and the higher amounts of epothilone C and D produced under a low oxygen tension.

[0062] The foregoing examples are for illustration of the present invention.

Claims

1. A method of modulating polyketide congener distribution production ratio in a myxobacterial host cell that comprises an active oxygen-dependent tailoring enzyme, the method comprising the steps of cultivating said host cell under depleted oxygen cultivation in growth medium under conditions that lead to the production of polyketides, wherein the production ratio of polyketide congeners is shifted from epothilone A and B to epothilone C and D.

2. The method of claim 1 further comprising the step of supplementing the growth medium with serine.

3. The method of claim 1 further comprising the step of supplementing the growth medium with acetate.

4. The method of claim 1 further comprising the step of supplementing the growth medium with propionate.

5. The method of claim 1 wherein said host cell is Myxococcus xanthus with an active EpoK⁺ monooxygenase.

6. The method of claim 1 wherein said host cell is Sorangium cellulosum.

7. The method of claim 1 wherein said host cell is Myxococcus xanthus strain K111.32.25, deposit no. PTA-1700.

8. The method of claim 1 wherein the oxygen-dependent tailoring enzyme is EpoK.

Ansprüche

1. Verfahren zur Modulierung des Polyketid-Congener-Verteilungsproduktionsverhältnisses in einer myxobakteriellen Wirtszelle, die ein aktives Sauerstoff-abhängiges tailoring-Enzym umfasst, wobei das Verfahren die Schritte der Kultivierung der Wirtszelle unter Sauerstoff-abgereicherter Kultivierung in Wachstumsmedium unter Bedingungen umfasst, die zur Produktion von Polyketiden führen, wobei das Produktionsverhältnis von Polyketid-Congenem von Epothilon A und B nach Epothilon C und D verschoben wird.

2. Verfahren nach Anspruch 1, das weiterhin den Schritt der Anreicherung des Wachstumsmediums mit Serin umfasst.

3. Verfahren nach Anspruch 1, das weiterhin den Schritt der Anreicherung des Wachstumsmediums mit Acetat umfasst.

4. Verfahren nach Anspruch 1, das weiterhin den Schritt der Anreicherung des Wachstumsmediums mit Propionat umfasst.

5. Verfahren nach Anspruch 1, wobei die Wirtszelle Myxococcus xanthus mit einer aktiven EpoK⁺ Monooxygenase ist.

6. Verfahren nach Anspruch 1, wobei die Wirtszelle Sorangium cellulosum ist.

7. Verfahren nach Anspruch 1, wobei die Wirtszelle Myxococcus xanthus, Stamm K111.32.25, Hinterlegungs-Nr. PTA-1700, ist.

8. Verfahren nach Anspruch 1, wobei das Sauerstoff-abhängige tailoring-Enzym EpoK ist.

Revendications

1. Procédé de modulation du rapport distribution/production de congénères de polycétides dans une cellule hôte myxobactérienne qui comprend une enzyme d'adaptation oxygénodépendante active, le procédé comprenant les étapes consistant à cultiver ladite cellule hôte par une culture appauvrie en oxygène dans un milieu de croissance dans des conditions qui débouchent sur la production de polycétides, dans lequel le rapport de production de congénères de polycétides est décalé des épothilones A et B vers les épothilones C et D.

2. Procédé selon la revendication 1, comprenant en outre l'étape consistant à supplémenter le milieu de croissance avec de la sérine.

3. Procédé selon la revendication 1, comprenant en outre l'étape consistant à supplémenter le milieu de croissance avec de l'acétate.

4. Procédé selon la revendication 1, comprenant en outre l'étape consistant à supplémenter le milieu de croissance avec du propionate.

5. Procédé selon la revendication 1, dans lequel ladite cellule hôte est Myxococcus xanthus avec une monooxygénase EpoK⁺ active.

6. Procédé selon la revendication 1, dans lequel ladite cellule hôte est Sorangium cellulosum.

7. Procédé selon la revendication 1, dans lequel ladite cellule hôte est la souche K111.32.25 de Myxococcus xanthus, numéro de dépôt PTA-1700.

8. Procédé selon la revendication 1, dans lequel l'enzyme d'adaptation oxygénodépendante est EpoK.

Drawing